Clinical epidemiology and high genetic diversity amongst Cryptococcus spp. isolates infecting people living with HIV in Kinshasa, Democratic Republic of Congo

Neuromeningeal cryptococcosis (NMC) is a life-threatening opportunistic infection in advanced HIV disease patients (AHDP). It is caused by Cryptococcus spp. complexes and mainly occurs in sub-Saharan Africa. In this study, we performed molecular characterization and antifungal susceptibility profiling of Cryptococcus isolates from AHDP in Kinshasa (DRC). Additionally, we investigated a possible association between NMC severity factors and the Cryptococcus neoformans (Cn) multilocus sequence typing (MLST) profiles. We characterized the isolates using PCR serotyping, MALDI-TOF MS, internal transcribed spacer (ITS) sequencing, and MLST. Susceptibility testing for the major antifungal drugs was performed according to the EUCAST guidelines. Parameters associated with NMC severity, such as hypoglycorrhachia (< 50 mg/dL), increased cerebral spinal fluid opening pressure (> 30 cm H2O), and poor therapeutic outcome were compared with the Cn MLST sequences type (ST). Twenty-three out of 29 Cryptococcus isolates were identified as serotype A using PCR serotyping (79.3%; 95% IC: 65.5–93.1), while six (20.7%; 95% IC: 6.9–34.5) were not serotypable. The 29 isolates were identified by ITS sequencing as follows: Cryptococcus neoformans (23/29, 79.3%), Cutaneotrichosporon curvatus (previously called Cryptococcus curvatus) (5/29, 17.2%), and Papiliotrema laurentii (Cryptococcus laurentii) (1/29, 3.5%). Using the ISHAM MLST scheme, all Cn isolates were identified as molecular type VNI. These comprised seven different STs: ST93 (n = 15), ST5 (n = 2), ST53 (n = 1), ST31 (n = 1), ST4 (n = 1), ST69 (n = 1), and one novel ST that has not yet been reported from other parts of the world and was subsequently assigned as ST659 (n = 2). Of the included strains, only Papiliotrema laurentii was resistant to amphoterin B (1/29, 3.5%), 6.8% (2/29) were resistant to 5-flucytosine (the single Papiliotrema laurentii strain and one Cryptococcus neoformans isolate), and 13.8% (4/29) to fluconazole, including two of five (40%) Cutaneotrichosporon curvatus and two of 23 (8.7%) C. neoformans strains. We found a significative association between poor therapeutic outcome and a non-ST93 sequence type of causative strains (these concerned the less common sequence types: ST53, ST31, ST5, ST4, ST659, and ST69) (87.5% versus 40%, p = 0.02). Molecular analysis of Cryptococcus spp. isolates showed a wide species diversity and genetic heterogenicity of Cn within the VNI molecular type. Furthermore, it is worrying that among included strains we found resistances to several of the commonly used antifungals.

In case of poor clinical condition or in minor patients, the approval of the guardian was obtained. In addition, all data were analysed anonymously.

and one novel ST identified 60
in the present work and subsequently assigned as ST659 (n=2). Among NMC severity factors, only the 61 patient pejorative outcome was associated with infections by less common STs isolates ( 7/8, 87.5%, 62 p=0.02) (ST53, ST31, ST5, ST4, ST659, and ST69). Molecular analysis of Cryptococcus spp. isolates 63 showed a wide species diversity and genetic heterogenicity of Cn within the VNI molecular type. 64 Furthermore, infections due to less common STs were associated with more pejorative outcomes than 65 those due to ST93.

Study design, patients and samples 100
A cross-sectional study was conducted in three Kinshasa public hospitals supported by Doctors without 101 Borders-Belgium (MSF), from 1 February 2019 to 29 February 2020. Thus, 278 patients were included 102 and among them, NMC was diagnosed based on the cryptococcal antigens (CrAg) detection among 103 patients and/or the presence of yeasts cells detected by India ink staining and/or by culture. 104

Biological analyses 105
The CrAg detection was carried out in the CSF of each included patient, using the CrAg LFA IMMY 106 test (Immuno-mycologic, Norman, OK, USA). Direct staining with India ink in the CSF was also carried 107 out, and the CSF was cultured on Sabouraud Dextrose Agar-Chloramphenicol medium (SDA-C, 108 bioMérieux, France) at 30°C for 48 to 72h. The qualitative test Pandy was performed for determining 109 proteinorachia as previously described [11].

DNA extraction 124
Genomic DNA was extracted from the fresh 24-hour cultures using the NucleoSpin blood quick pure 125 kit (Macherey-Nagel, Düren, Germany). Two preliminary steps were added to the manufacturer's 126 protocol, namely bead-beating and thermal shock. In a 2mL tube containing 0.5mm glass beads (Roche 127 Diagnostics GmbH, Penzberg, Germany), colonies were mixed with 350µL lysis buffer (Promega 128 Corporation, USA). The mixture was vortexed five times due to 6000 vibrations per minute for 40 129 seconds (bead-beating). Between each pass, the tube was cooled between -20°C and 1°C for 30 seconds 130 in a Nalgene microtube cooler container (Dutscher, France) for a thermal shock.

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A classical serotyping PCR designed for Cn/Cg species complex was performed according to the 133 protocol described by Ito-Kuwa et al. [13]. 134

ITS sequencing 135
The ITS2 region of the rRNA gene cluster was amplified using the ITS86 forward primer 136 5′GTGAATCATCGAATCTTTGAA 3′ and ITS4 reverse primer 5′TCCTCCGCTTATTGATATGC 3′ 137 [14]. The amplified products were purified using the kit clean Seq Agencourt (Beckman Coulter Life 138 Science). The sequencing was done on the automate ABI 3500/3500XL (Applied Biosystem, Life 139 Technologies). Bidirectional sequence data were generated after purification using the BigDye Only results that repeated the same identification at least three times and had a similarity score greater 145 than 95% were considered valid. 146

Multilocus sequence typing (MLST) was performed using the International Society of Human and 148
Animal Mycology (ISHAM) consensus scheme for the Cn/Cg species complexes; including six unlinked 149 housekeeping loci (GPD1, LAC1, URA5, SOD1, CAP59, and PLB1) and the non-coding region IGS1 150 [6]. After DNA extraction, samples were sequenced using Illumina HiSeq as previously described [15], 151 and the raw contigs sequences were paired, removed of duplicate reads, and trimmed using Geneious 152 Prime 64_2021_1 (https://www.geneious.com). Then, the MLST loci were extracted by mapping to the 153 reference sequences of each MLST locus from the online ISHAM MLST fungal database 154 (https://mlst.mycologylab.org/) and each allele type (AT) was assigned using the same online database. 155 The ATs combination defined the sequences type (ST) which in most cases corresponds to the species 156 Molecular type. 157

Phylogenetic analysis 158
Phylogenetic analysis of concatenated sequences of the seven MLST loci was performed using MEGA 159 v.6.06 software (http://www.ebi.ac.uk/tools/msa/clustalo). A dendrogram was produced by the 160 Maximum Likelihood method using sequences alignment with the Kimura 2-parameter method. Gaps 161 were treated as a complete deletion. Statistical support for each clade was assessed using bootstrap 162 analysis with 1000 replicates. Apart from C. neoformans and C. gattii reference strains (WM) included 163 in the analysis, the MLST sequences of the only C. neoformans strain (ZS) previously isolated from 164 Congolese infected patient (DRC) was also included. 165

Statistical analysis
The analysis was carried out using R-cmdr version 2.6-1 (R Foundation for Statistical Computing, 167 Vienna, Austria). Missing data were considered completely random and the available data were 168 analyzed. The continuous variables were summarised as mean ± standard deviation and compared using 169 Student's t-test. The proportions and their respective 95% confidence intervals were calculated for the 170 categorical data. The main outcome variable was the NMC diagnosis. This variable was compared to 171 other variables of the same category using Pearson's chi-square test or Fisher's exact test if the expected 172 values were less than five. Very raised CSF opening pressure (>30cm water), hypoglycorrhachia 173 (<50mg/dL) and patients' pejorative outcome were considered in performing the association analysis 174 between the NMC severity factors and the identified ST-MLST profile. Also, two isolates categories 175 were formed according to the STs-MLST profile: the main ST isolated on the one hand, and the other 176 STs on the other hand. All tests were two-tailed and a p < 0.05 was considered statistically significant. 177

Ethical considerations 181
This work was carried out in strict compliance with ethical rules, with the approval of the Ethics 182   Table 2. 217

MLST result 220
Apart from the six strains identified as C. curvatus or C. laurentii, the remaining 23 C. neoformans    Plus, macrophages from women phagocytized more C. neoformans than those from men. The men 276 hence had a higher fungal load than women and their macrophages were more likely to be destroyed by 277 C. neoformans [19]. This protective trend in women was not significantly noted in the current study. 278 Headache, convulsions and visual disturbances were significantly associated with NMC. This data is 279 largely consistent with the literature [20]. Slightly more than the proportion reported in this study (7.7%), 280 visual disturbances are known to be associated with NMC in 18% of cases following raised intracranial 281 pressure [21]. Among the clinical parameters (headache, sensorium depression, papilledema, and raised 282 CSF opening pressure) and the radiological ones (flattening of the posterior sclera, increased CSF in the 283 subarachnoid space around the optic nerve, optic nerve tortuosity and empty parietal saddle) defining 284 these disorders, only headaches and raised CSF opening pressure were found in the present study [21]. 285 NMC was associated with higher raised CSF opening pressure, CD4 count < 100 cells/mm 3 and HIV-286 infection stage IV. One of the most critical outcome determinants in PLHIV with NMC is the raised 287 opening CSF pressure which is generally correlated with high CSF fungal load, morbidities and 288 increased risk of death [22]. In agreement with the data described by Bicanic  Cryptococcus India ink staining identification were very low in the present study [24]. For Kabanda T.  neoformans was identified for 73.3% of all isolates. The remaining cases were identified as C. curvatus 299 (17.2%) and C. laurentii (3.5%). Initially considered saprophytic and non-pathogenic to humans, the 300 non-neoformans and non-gattii Cryptococcus species are increasingly found in clinical infections in 301 recent years [8]. Although the C. neoformans NMC clinical presentation is more severe in PLHIV than 302 in non-neoformans/ non-gattii NMC, C. curvatus and C. laurentii have a high tendency to be fluconazole 303 and 5-flucytosine resistant. In addition, these two latter species are more difficult to identify by routine 304 laboratory methods than C. neoformans [8]. Thus, out of six non-neoformans and non-gattii isolates, 305 only four were correctly identified by MALDI-TOF MS and confirmed by ITS sequencing. The lack of 306 certain species reference spectra in the database provided by the mass spectrometry manufacturer 307 (Bruker: BD 83) for identification, and genome differences between Cryptococcus species, could 308 explain these results. Worldwide, serotype A isolates remains the most commonly isolated in 309 environmental and clinical settings [28], a trend also observed in the current study. 310 The MLST analysis of C. neoformans isolates revealed large heterogeneity of STs within the single 311 molecular type found in the present study (VNI), involving seven distinct STs. In line with our results, 312 Cryptococcus neoformans ST93 is the most isolated in various countries (China, India, Indonesia, South 313 Africa, Thailand, Brazil, Uganda, and Colombia), both in clinical and environmental settings. It has 314 been associated with high mortality in Uganda [29]. In this study, ST93 was associated with a less 315 pejorative treatment outcome than the less common STs. It hence opens up the debate on the relative 316 virulence of each ST compared to the others. Although most of the STs had already been isolated in the 317 DRC neighbouring countries, namely ST4 in Uganda and Tanzania, ST5, ST31, ST69, and ST93 in 318 Uganda only, one ST (ST53) had previously been isolated only in Thailand and in no other country 319 worldwide (https://mlst.mycologylab.org/). Plus, one isolate had an ST identified for the first time in the 320 present study, subsequently assigned as ST659. The Congolese strain (ZS CN ST32) isolated 30 years 321 earlier is closely related to the isolates of the dominant ST (ST93) identified in the current study. 322 As described by Trilles et al. regarding the low antifungal susceptibility of VGI isolates compared to 323 the other molecular types tested [30], the less common STs isolates identified in the current study were 324 associated with poor therapeutic outcomes. 325

CONCLUSIONS 326
A more severe epidemiological profile of NMC than previously reported from the DRC was found using 327 a panel of diagnostic tests in symptomatic PLHIV. Apart from the species diversity identified amongst 328 the yeasts isolated from CSF samples, including C. neoformans, C. curvatus and C. laurentii, the STs 329 within the single molecular type VNI identified in the present study showed great heterogeneity, 330 including seven different STs with one major ST (ST93) and six less common STs (ST5, ST659, ST53, 331 ST31, ST4, and ST69). In addition, the less common STs isolates were associated with the patient 332 pejorative outcomes. More robust studies including larger sampling numbers and antifungal 333 susceptibility of isolates could improve the understanding of the data from this study.